Mx3005p qpcr instrument

A standard curve of the P. infestans 1004-bp P. infestans fragment was established as described above in the DNA dilution solution. The reactions were performed in a 25 μL final volume containing 5 μL of DNA extract, 12.5 μL of 2x QuantiFast Multiplex Probe PCR Master Mix (Qiagen Inc, Mississauga, ON, Canada), 300 nM each of primers ITS2HR-pinf-F, ITS2HR-pinf-R, EIPC100-F and EIPC100-R and 200 nM each of TaqMan probes ITS2HR-pinf-P and EIPC100-P. The two-step PCR conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 30 s and 62°C for 30 s in a Mx3005P qPCR Instrument (Agilent Technologies, Santa Clara, CA, USA). The results were evaluated for the efficiency of P. infestans amplification and the stability of EIPC detection among the dilutions.

The tetramethylrhodamine (TAMRA)/disulfide-labeled DNA hairpin S4 (1 μL, 1 mM in H₂O, 1 nmol) was thermally annealed with the complementary biotinylated strand S5 (1.1 μL, 1.1 mM in H₂O, 1.1 nmol) in phosphate buffer solution (PBS, 10 μL, 0.1 M, pH 7.0). The disulfide group was reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 0.5 μL, 0.5 M in H₂O) at 18°C for 1.25 h. Then, the resulting double stranded DNA (dsDNA) was conjugated to streptavidin-coated magnetic nanoparticles (Dynabeads, Thermofisher), and transferred into Gly_0.07 (200 μL). The maleimide ester S21 (1 mg, 2.5 μmol) was added to the solution and allowed to react for 2 h. The nanoparticle-supported DNA-conjugated activated ester was transferred into clean Gly_0.07 and the hairpin was released by toehold-mediated strand exchange by addition of one equivalent of strand S6 at 18°C for 2 h to produce an approximately 5 μM solution of 4. Finally, the solution of 4 (5 μL) was diluted in the DES of interest (95 μL), the resulting solution briefly centrifuged, and the fluorescein (FAM) fluorescence recorded over 24 h in a Mx3005P qPCR instrument (Agilent). The spectral overlap between FAM fluorophore (S21) and TAMRA-labeled DNA (S4) was also determined (Figure S4).

Analysis of the thermal stability of recombinant VgrG2b_C-ter protein in the presence and absence of zinc ions was determined using a Mx3005P qPCR instrument (Agilent). VgrG2b_C-ter was used at 1.25 μM, in a buffer of 10 mM HEPES pH 7.4 and 100 mM NaCl supplemented with zinc acetate as indicated. The fluorescent dye SyproOrange (Sigma Aldrich) was used at a 1/1000 dilution and the thermal unfolding of VgrG2b_C-ter was monitored between 25-98°C at a rate of 1.5K/min. Excitation occurred at 492 nm and emission fluorescence at 610 nm was measured every 40 s. Non-linear least-squares fitting (Kemmer and Keller, 2010 (link)) was used to analyze the raw data and the melting point of each sample was determined from the inflection point of the fitted curve.

For reverse transcription, 200-300ng of total RNA was used. The reaction was performed using MultiScribe reverse transcriptase (Thermo Fisher Scientific), random hexamers and RNAase inhibitor (both Roche). Subsequently, the qPCR reaction was performed using the SYBR green GoTaq qPCR master mix (Promega) on Agilent Stratagene Mx3005P qPCR instrument. Sequences of primers are listed in Supplementary Table 1. Relative gene expression was calculated using the 2^-ΔΔCt method. GAPDH was used as the reference gene.